Expression of a Recombinant High Affinity IgG Fc Receptor by Engineered NK Cells as a Docking Platform for Therapeutic mAbs to Target Cancer Cells.

Anti-tumor mAbs are the most widely used and characterized cancer immunotherapy. Despite having a significant impact on some malignancies, most cancer patients respond poorly or develop resistance to this therapy. A known mechanism of action of these therapeutic mAbs is antibody-dependent cell-mediated cytotoxicity (ADCC), a key effector function of human NK cells. CD16A on human NK cells has an exclusive role in binding to tumor-bound IgG antibodies. Though CD16A is a potent activating receptor, it is also a low affinity IgG Fc receptor (FcγR) that undergoes a rapid downregulation in expression by a proteolytic process involving ADAM17 upon NK cell activation. These regulatory processes are likely to limit the efficacy of tumor-targeting therapeutic mAbs in the tumor environment. We sought to enhance NK cell binding to anti-tumor mAbs by engineering these cells with a recombinant FcγR consisting of the extracellular region of CD64, the highest affinity FcγR expressed by leukocytes, and the transmembrane and cytoplasmic regions of CD16A. This novel recombinant FcγR (CD64/16A) was expressed in the human NK cell line NK92 and in induced pluripotent stem cells from which primary NK cells were derived. CD64/16A lacked the ADAM17 cleavage region in CD16A and it was not rapidly downregulated in expression following NK cell activation during ADCC. CD64/16A on NK cells facilitated conjugation to antibody-treated tumor cells, ADCC, and cytokine production, demonstrating functional activity by its two components. Unlike NK cells expressing CD16A, CD64/16A captured soluble therapeutic mAbs and the modified NK cells mediated tumor cell killing. Hence, CD64/16A could potentially be used as a docking platform on engineered NK cells for therapeutic mAbs and IgG Fc chimeric proteins, allowing for switchable targeting elements and a novel cancer cellular therapy.

A novel, long-lived, and highly engraftable immunodeficient mouse model of mucopolysaccharidosis type I.

Mucopolysaccharidosis type I (MPS I) is an inherited α-L-iduronidase (IDUA, I) deficiency in which glycosaminoglycan (GAG) accumulation causes progressive multisystem organ dysfunction, neurological impairment, and death. Current MPS I mouse models, based on a NOD/SCID (NS) background, are short-lived, providing a very narrow window to assess the long-term efficacy of therapeutic interventions. They also develop thymic lymphomas, making the assessment of potential tumorigenicity of human stem cell transplantation problematic. We therefore developed a new MPS I model based on a NOD/SCID/Il2rγ (NSG) background. This model lives longer than 1 year and is tumor-free during that time. NSG MPS I (NSGI) mice exhibit the typical phenotypic features of MPS I including coarsened fur and facial features, reduced/abnormal gait, kyphosis, and corneal clouding. IDUA is undetectable in all tissues examined while GAG levels are dramatically higher in most tissues. NSGI brain shows a significant inflammatory response and prominent gliosis. Neurological MPS I manifestations are evidenced by impaired performance in behavioral tests. Human neural and hematopoietic stem cells were found to readily engraft, with human cells detectable for at least 1 year posttransplantation. This new MPS I model is thus suitable for preclinical testing of novel pluripotent stem cell-based therapy approaches.

Glycogen synthase kinase 3 (GSK3) inhibitor, SB-216763, promotes pluripotency in mouse embryonic stem cells.

Canonical Wnt/ß-catenin signaling has been suggested to promote self-renewal of pluripotent mouse and human embryonic stem cells. Here, we show that SB-216763, a glycogen synthase kinase-3 (GSK3) inhibitor, can maintain mouse embryonic stem cells (mESCs) in a pluripotent state in the absence of exogenous leukemia inhibitory factor (LIF) when cultured on mouse embryonic fibroblasts (MEFs). MESCs maintained with SB-216763 for one month were morphologically indistinguishable from LIF-treated mESCs and expressed pluripotent-specific genes Oct4, Sox2, and Nanog. Furthermore, Nanog immunostaining was more homogenous in SB-216763-treated colonies compared to LIF. Embryoid bodies (EBs) prepared from these mESCs expressed early-stage markers for all three germ layers, and could efficiently differentiate into cardiac-like cells and MAP2-immunoreactive neurons. To our knowledge, SB-216763 is the first GSK3 inhibitor that can promote self-renewal of mESC co-cultured with MEFs for more than two months.